GCN4 is a transcriptional activator in S. cerevisiae that is regulated at the translational level by upstream open reading frames (uORFs) in the GCN4 mRNA leader. In amino acid-starved cells, ribosomes translate uORF1, bypass the remaining uORFs 2-4, and reinitiate at GCN4 instead because they fail to bind the eIF-2(initiation factor-2)~GTP~tRNA(Met)i ternary complex. The level of ternary complexes is reduced in starved cells by phosphorylation of the alpha subunit of eIF-2 by GCN2. In mammalian cells, phosphorylated eIF-2 sequesters eIF-2B, the eIF-2-guanine nucleotide exchange factor. GCD1, GCD2, GCD6, GCD7 and GCN3, translational regulators of GCN4, were shown to be components of the eIF-2B in yeast. Mutations in eIF-2alpha, GCN3, GCD7 and GCD2 were isolated that suppress the toxic effects of eIF-2alpha hyperphosphorylation by GCN2c proteins, indicating that eIF-2 phosphorylation stimulates GCN4 translation by impairing eIF-2B. Deletions of GCN1 and GCN20 also suppress GCN2c alleles and the GCN1 and GCN20 proteins are required for GCN2-catalyzed phosphorylation of eIF-2alpha in vivo, probably functioning to couple GCN2 kinase activity to uncharged tRNA. Suppressors of GCN2c alleles affecting subunits of eIF-2 or eIF-2B overcome the toxic effects of expressing the mammalian eIF-2alpha kinases DAI and HRI in yeast, whereas GCN1 mutations only suppress GCN2c alleles, indicating that GCN1 is a positive regulator of GCN2 and not a negative regulator of an eIF-2alpha phosphatase. DAI is activated by double-stranded-RNA (dsRNA) during viral infections in mammalian cells, and translational control by DAI in yeast was shown to be dependent on amino acids in its regulatory domain that are conserved among dsRNA-binding proteins; thus, dsRNA-binding activates DAI kinase function in vivo. Purine starvation activates GCN2 in yeast and this response is required for wild-type purine biosynthesis and transcription of ADE8 under conditions of purine limitation. Genetic analysis revealed the existence of at least two non-overlapping activation domains in GCN4 that, to a first approximation, are functionally redundant. GCN4 proteins lacking one of these domains are toxic, reflecting the sequestration of a general transcription factor or repression of an essential gene. Chromosomal mutations were isolated that restore transcriptional activation by a gcn4 allele containing only an incomplete copy of one activation domain. We have also characterized mutations that alter transcriptional regulation of a ribosomal protein gene according to amino acid availability.